Lectins are proteins derived from plant, animal, or microbial sources which have specific carbohydrate-binding activity. Because of their binding specificity for sugars and oligosaccharides, lectins have become useful reagents in the study of the biology and biochemistry of glycoconjugates in vitro (Rhodes et al., 1988, Digest. Dis. Sci., 33:1359-1363; and Green and Baenziger, 1989, Trends Biochem Sci., 14:168-171), and for the analysis of cellular components of the mammalian immune system (Smith, 1972, Transplant Rev., 11:179-216; and MacDonald and Natoholz, 1986, Ann. Rev. Cell. Biol., 2:231-254). The interaction of lectins with surface g1Ycoproteins of various cells of the immune system induce or inhibit a variety of responses in vitro which are indicative of cellular functions in vivo. For example, lectins can induce mitogenesis of T-lymphocytes (Favero et al., 1988, Cell. Immunol., 142:4401-4406) and induce synthesis and secretion of a variety of cytokines which are proteins involved in the regulation of immune responses (Miyajima et al., 1988, FASEB J., 2:2462-2473; and Taniguchi, 1988, Ann. Rev. Immunol. 6:439-464).
Many lectins have the potential for being powerful therapeutic and diagnostic reagents in vivo due to their carbohydrate-binding activity and specificity. Numerous lectins have been identified which could be screened for these purposes (Sharon and Lis, 1987, Tends Biochem. Sci. 12:488-491). However, principal factors which limit the development of lectins for in vivo use include their inherent toxicity, their potential to elicit an immunogenic response, and the short half-life of these substances in the circulatory system. In particular, an immune response against the lectins would enhance the destruction and clearance of subsequent introductions of the lectin, in addition to the possibility of causing an allergic reaction.
An inert water-soluble polymer may be conjugated to proteins to alter their properties (Inada et al. 1988, Trends Biotech. 6:131-134). The reduction in toxicity, immunogenicity, and clearance rate of certain polymer-protein conjugates has led to using polymer conjugation as a drug delivery system (Krieger, 1990, Chem. Eng. News, 68:38-40). For example, U.S. Pat. No. 4,179,337, discloses a process for coupling polypeptides, including enzymes and hormones, to polymers such as PEG. The resulting PEG-polypeptide conjugates are substantially non-immunogenic and retain some of the desired physiological activity of the base polypeptide when injected into the mammalian circulatory system.
U.S. Pat. No. 4,791,192 discloses a chemically modified protein which comprises an islet-activating protein produced by bacteria belonging to the genus Bordetella and a polyethylene glycol moiety bound with a primary amino group of the islet-activating protein.
U.S. Pat. No. 4,496,689, discloses a process for producing covalent conjugates formed by the chemical coupling reaction of the blood plasma glycoprotein, alpha-1-proteinase inhibitor, with an "activated" water soluble polymer, such as PEG. The conjugates are used in a method for treating pulmonary emphysema and respiratory distress syndrome.
Although conjugation of proteins to water-soluble polymers has been described in the prior art, it cannot be expected that the carbohydrate-binding activity of lectins can be retained after coupling because of potential steric hindrances involved in conjugation. Typically modification, such as by conjugation, of lectins involves covalent coupling between constituents of one or more functional groups of the modifying agent and one or more amino acids of the particular lectin. Thus, modification of lectins can result in concomitant loss of bioactivity, i.e. carbohydrate-binding activity, when amino acid residues of the lectin affected by the modification are amino acids which are necessary for bioactivity. The prior art fails to disclose a method which overcomes the problem of loss of carbohydrate-binding activity from modification of the lectin. Therefore, there exists a need for a method for predictably producing a water-soluble polymer-lectin conjugate that retains carbohydrate-binding activity subsequent to conjugation.
Additionally, lectins have been unable to achieve their potential for being biological response modifier in vivo because of their toxicity, immunogenicity and rapid clearance. Therefore, a need exists for carbohydrate-binding lectin derivatives which may be useful as therapeutic and diagnostic reagents in vivo and have reduced toxicity and immunogenicity with extended biologically active lifetime in vivo.